Figure 1.
Endothelial cells in low and high glucose medium did not align in the flow direction, whereas cells in normal glucose aligned.
Cells in high glucose also aligned in response to cyclic strain. Actin (red) and nuclei (blue) before and after A) shear stress and B) cyclic strain, both applied in the horizontal direction. Scale bar = 50 µm. C) Percentage aligned actin fibers for shear stress, D) average actin fiber angle (absolute value) for shear stress, and E) percentage aligned actin fibers for cyclic strain. *p<0.01, #p<0.05 compared to static sample for the same culture condition. Shear stress experiments were completed in duplicate, and cyclic strain experiments were completed in triplicate. Each experiment was repeated three times.
Figure 2.
eNOS phosphorylation increased with shear stress for endothelial cells in normal glucose but did not significantly change for cells in low or high glucose.
A) p-eNOS (magenta) and nuclei (blue) after 30 seconds shear stress (horizontal direction). Scale bar = 50 µm. B) p-eNOS mean fluorescence intensity. * p<0.01 compared to static sample for the same glucose condition. Experiments were completed in duplicate and repeated three times.
Figure 3.
PAEC in low and high glucose did not activate FAK in response to shear stress, whereas shear stress did increase phosphorylated Akt in high glucose cells.
A) p-FAK (magenta) and nuclei (blue) after 30 seconds shear stress (horizontal direction). Scale bar = 50 µm. p-FAK mean fluorescence intensity. *p<0.01 compared to static sample for the same glucose condition. B) Akt phosphorylation after 30 minutes of shear stress. p-Akt normalized to total Akt. *p<0.01, #p<0.05 compared to static sample for the same glucose condition. Experiments were completed in duplicate and repeated three times.
Figure 4.
ROS and PKC were elevated in both low and high glucose.
A) carboxy-H2DCFDA (ROS, green) and C) Fim-1 diacetate (PKC, yellow) after 48 hours in different glucose conditions. Scale bar = 50 µm. B) ROS and D) PKC fluorescence mean fluorescence intensity. #p<0.05 compared to NG cells. Experiments were completed in triplicate and repeated three times.
Figure 5.
PKC blockade restored FAK phosphorylation and actin alignment in response to shear stress in cells cultured in high glucose.
A) p-FAK (magenta) and nuclei (blue) with 2 hours Fim-1 diacetate (200 nM) followed by 30 seconds shear stress (horizontal direction). Scale bar = 50 µm. C) p-FAK mean fluorescence intensity. * p<0.01 compared to static. B) Actin (red) and nuclei (blue) for high glucose cells with PKC blocked by 200nM Fim-1 diacetate and 200 nM chelerythrine, and normal glucose cells with PKC activated by 1 µM PMA. Shear stress was applied for 12 hours (horizontal direction). Scale bar = 50 µm. D) and E) Actin aligned fiber percentage and average angle absolute value. * p<0.01 compared to static sample for the same culture condition. Experiments were completed in duplicate and repeated three times.
Figure 6.
VEGF secreted by endothelial cells in low glucose translocated β-catenin at cell-cell junctions to the nucleus.
A) VEGF in conditioned medium after two days. * p<0.01 compared to NG cells. B) β-catenin (green) after VEGF treatment (100 ng/mL) of PAEC in normal glucose.* p<0.01 compared to 0 minutes VEGF exposure. C) β-catenin (green) in PAEC in low glucose. Scale bar = 50 µm. * p<0.01 compared to NG cells. Experiments were completed in triplicate and repeated two times.
Figure 7.
Actin fibers aligned in low glucose with shear stress when VEGF was blocked, and VEGF addition to cells in normal glucose prevented actin alignment with shear stress.
A) Actin (red) and nuclei (blue) in low glucose cells with VEGF blocked for 24 hours with a neutralizing antibody (1 µg/ml) or normal glucose cells with VEGF added (50 ng/mL) and exposed to 12 hours shear stress. Scale bar = 50 µm. B) Actin fiber percentage and average actin fiber angle (absolute value). *p<0.01 compared to static sample for the same culture condition. Experiments were completed in duplicate and repeated three times.
Figure 8.
Proposed signaling pathways in low and high glucose.
A) Endothelial cells in low glucose release VEGF, which disrupts β-catenin at adherens junctions. This then impairs mechanosensory complex activation due to shear stress, inhibiting downstream responses. B) Cells in normal glucose sense shear stress at the mechanosensory complex, phosphorylate eNOS, and align actin. C) Cells in high glucose maintain an intact mechanosensory complex, however elevated PKC inhibits FAK activation and subsequent actin alignment.